High-throughput imaging of ATG9A distribution as a diagnostic functional assay for adaptor protein complex 4-associated hereditary spastic paraplegia
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Authors
Alecu, JE
Brechmann, B
Ziegler, M
Eberhardt, K
Jumo, H
D'Amore, A
Faghihi, MA
De Bleecker, JL
Vuillaumier-Barrot, S
Santorelli, FM
Neuser, S
Barrett, L
Davies, AK
Saffari, A
Hirst, J
Sahin, M
Publication Date
2021Journal Title
Brain Communications
ISSN
0364-5134
Volume
3
Issue
4
Language
eng
Type
Article
This Version
VoR
Metadata
Show full item recordCitation
Ebrahimi-Fakhari, D., Alecu, J., Brechmann, B., Ziegler, M., Eberhardt, K., Jumo, H., D'Amore, A., et al. (2021). High-throughput imaging of ATG9A distribution as a diagnostic functional assay for adaptor protein complex 4-associated hereditary spastic paraplegia. Brain Communications, 3 (4) https://doi.org/10.1093/braincomms/fcab221
Abstract
Adaptor protein complex 4-associated hereditary spastic paraplegia is caused by biallelic loss-of-function variants in <i>AP4B1</i>, <i>AP4M1</i>, <i>AP4E1</i> or <i>AP4S1</i>, which constitute the four subunits of this obligate complex. While the diagnosis of adaptor protein complex 4-associated hereditary spastic paraplegia relies on molecular testing, the interpretation of novel missense variants remains challenging. Here, we address this diagnostic gap by using patient-derived fibroblasts to establish a functional assay that measures the subcellular localization of ATG9A, a transmembrane protein that is sorted by adaptor protein complex 4. Using automated high-throughput microscopy, we determine the ratio of the ATG9A fluorescence in the trans-Golgi-network versus cytoplasm and ascertain that this metric meets standards for screening assays (Z'-factor robust >0.3, strictly standardized mean difference >3). The 'ATG9A ratio' is increased in fibroblasts of 18 well-characterized adaptor protein complex 4-associated hereditary spastic paraplegia patients [mean: 1.54 ± 0.13 versus 1.21 ± 0.05 (standard deviation) in controls] and receiver-operating characteristic analysis demonstrates robust diagnostic power (area under the curve: 0.85, 95% confidence interval: 0.849-0.852). Using fibroblasts from two individuals with atypical clinical features and novel biallelic missense variants of unknown significance in <i>AP4B1</i>, we show that our assay can reliably detect adaptor protein complex 4 function. Our findings establish the 'ATG9A ratio' as a diagnostic marker of adaptor protein complex 4-associated hereditary spastic paraplegia.
Keywords
Biomarker, Hereditary Spastic Paraplegia, Functional Assay, High-throughput Imaging, Adaptor Protein Complex 4
Sponsorship
Wellcome Trust (086598, 100140, 214272)
Identifiers
PMC8557665, 34729478
External DOI: https://doi.org/10.1093/braincomms/fcab221
This record's URL: https://www.repository.cam.ac.uk/handle/1810/332224
Rights
Attribution-NonCommercial 4.0 International
Licence URL: https://creativecommons.org/licenses/by-nc/4.0/
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